Identification of cell wall deficient forms of M-avium subsp paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization

    Authors

    K. Hulten; T. J. Karttunen; H. M. T. El-Zimaity; S. A. Naser; M. T. Collins; D. Y. Graham;F. A. K. El-Zaatari

    Abbreviated Journal Title

    J. Microbiol. Methods

    Keywords

    in situ hybridization; cell wall deficient (CWD); mycobacteria; Johne's; disease; Crohn's disease; POLYMERASE CHAIN-REACTION; DETECT MYCOBACTERIUM-PARATUBERCULOSIS; CROHNS-DISEASE; INSITU HYBRIDIZATION; DNA; SARCOIDOSIS; PYLORI; PROBES; IS900; COWS; Biochemical Research Methods; Microbiology

    Abstract

    M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease (JD) in ruminants leading to enormous economical losses in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although only considered an animal pathogen, cell wall deficient (CWD) forms of M. paratuberculosis have been isolated from patients with sarcoidosis and Crohn's disease (idiopathic diseases) in humans. Hence, the CWD form of this organism has been suspected to play a role in the pathogenesis of these diseases by persisting in the affected tissues and triggering a localized immune response and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is the pathogenic form in the subclinical cases of JD in animals and/or the etiologic agent for the above human diseases. To localize such organisms in tissue sections, CWD forms of mycobacteria were prepared in vitro and injected into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuberculosis-specific probe labeled with digoxigenin, was developed for the detection of nucleic acids specifically from the CWD forms but not their acid-fast forms in tissue sections. Specificity was confirmed by the negative finding with an irrelevant probe and with control tissue preparations containing CWD cells of related mycobacteria and unrelated organisms. This ISH procedure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn's disease and sarcoidosis. (C) 2000 Elsevier Science BN. All rights reserved.

    Journal Title

    Journal of Microbiological Methods

    Volume

    42

    Issue/Number

    2

    Publication Date

    1-1-2000

    Document Type

    Article

    Language

    English

    First Page

    185

    Last Page

    195

    WOS Identifier

    WOS:000089797300009

    ISSN

    0167-7012

    Share

    COinS